We are continuing to make progress in our goal of defining germline modifiers of tumor progression and metastasis in PC. We have made extensive use of the well characterized C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of aggressive NEPC to investigate the role of hereditary factors in the development of aggressive PC. Our earlier work conclusively proved that the introduction of germline polymorphism into this model by breeding significantly modulates tumor progression and metastasis. Specifically, by crossing TRAMP mice to the eight progenitor strains of the Collaborative Cross (CC) recombinant inbred panel we observed profound differences in tumor burden and metastasis frequencies in several TRAMP x CC progenitor F1 strains. In the last year, we have used a QTL mapping approach to identify loci driving susceptibility to aggressive disease development in the TRAMP mouse model. The major focus of our ongoing work focuses on the use of the DO heterogeneous stock mice, which are a highly diverse mapping resource that are genetically related to the CC. DO mice are bred to the TRAMP model of NEPC to introduce genetic variation through breeding. Thus far, we have aged and phenotyped a population of approximately 500 (TRAMP x DO) F1 males and preliminary QTL analyses have identified multiple novel loci associated with aggressive disease development. These analyses revealed two QTLs associated with metastasis that achieved genome-wide significance, on mouse chromosomes 8 and 15. For the chromosome 8 locus, analysis of strain-specific allelic effects indicated that linkage was driven by the 129S1/SvlmJ and PWK/PhJ strains; and for the chromosome 15 locus, linkage was driven by the PWK/PhJ, NZO/HILtJ and WSB/EiJ strains. Trait-correlation and expression QTL data derived from microarray analysis of 90 (TRAMP x DO) F1 tumors were integrated with strain-specific SNP data to identify 24 candidate metastasis susceptibility genes. The role of these genes in aggressive human PC was investigated via an in silico validation that utilized human tumor gene expression datasets such as The Cancer Genome Atlas prostate adenocarcinoma dataset, and a human PC genome-wide association study (GWAS) cohort consisting of 1,172 PC patients. This approach led to the identification of 8 novel aggressive PC susceptibility genes. Ongoing work is focusing on the functional characterization of these genes to more fully comprehend their role in susceptibility to metastasis in PC. Additionally, we are performing RNA-seq analysis of primary tumors and normal prostates derived from (TRAMP x DO) F1 males to more fully comprehend the transcriptional landscape seen in this mouse model, and how it relates to aggressive human PC. Additionally, in the last year we have completed F2 intercross mapping studies using the TRAMP mouse and two of the CC progenitor strains that displayed the greatest phenotypic variation at the F1 generation. First, we have identified CXCL14, ITGAX and LPCAT2 as novel aggressive PC susceptibility genes in an F2 intercross involving TRAMP and the NOD/ShiLtJ strain. QTL mapping was performed in transgene-positive (TRAMP x NOD/ShiLtJ) F2 intercross males (n = 228), which facilitated identification of 11 loci associated with aggressive disease development. Microarray data derived from 126 (TRAMP x NOD/ShiLtJ) F2 primary tumors were used to prioritize candidate genes within QTLs, with candidate genes deemed as being high priority when possessing both high levels of expression-trait correlation and a cis-expression QTL. This process enabled the identification of 27 aggressive prostate tumorigenesis candidate genes. The role of these genes in aggressive forms of human PC was investigated by integrating patient outcome data from human PC tumor gene expression datasets with candidate gene polymorphism data from a human PC GWAS. This study is the first published example of using a systems genetics approach to successfully identify novel susceptibility genes for aggressive PC. Second, a similar approach using data derived from the (TRAMP x PWK/PhJ) F2 intercross population has allowed for the identification of GNL3 and SKA3 as novel germline metastasis susceptibility genes. As with the (TRAMP x NOD/ShiLtJ) F2 intercross, the role of each of these genes is being examined in aggressive human PC through a combination of expression profiling in human prostate tumors and analysis of human PC GWAS datasets. Functional analysis of the candidate genes identified in these F2 intercross studies is currently ongoing in PC cell lines and patient-derived tissues. Finally, we are utilizing the Hi-Myc mouse model of prostate tumorigenesis, which represents a much more indolent form of prostate adenocarcinoma. Specifically, we are investigating how genetic variation enhances the propensity for aggressive PC development by crossing Hi-Myc females with DO males. Thus far, we have aged and phenotype a cohort of 156 transgene-positive (Hi-Myc x DO) F1 males. Significantly, we have observed pulmonary metastasis in >12% of these animals, which is of significance since metastasis has never been described in this mouse model of PC. QTL mapping studies will aim to identify the modifier genes increasing susceptibility to metastasis in the Hi-Myc mouse model, and the relevance of these to aggressive human PC.